Vitamin b12



Patented Aug. 7, 1951 UNITED". srArss PATENT. OFFICE VITAMIN B12 Edward L.- Rickes, Union, and Thomas R. Wood, Murray Hill, N. J., assignors to Merck & 00., Inc., Railway, N. J a corporation of New Jersey No Drawing. Application August 4, 1949, Serial No. 108,668

' growth of Lactobacillus lactis Dorner, and possesses marked and effective action in the therapeutic treatment of Addisonian pernicious anemia and other macrocytic anemias.

Regarded in certain of its broader aspects, the process according to the present invention, comprises extracting a crude concentrate predominantly composed of Streptomyces griseus elaboration products with a solvent, effecting a chromatographic fractionation by adding said solvent solution to a column of adsorbent material, washing the column with solvent to fractionally elute the active substance from the adsorbent medium and recovering crystalline vitamin B12 from the eluate.

In preparing our novel chemical compounds, we utilize as a starting material a crude L. L. D. active concentrate of Streptomyces griseus elaboration products containing in some instances the antibiotic streptomycin or grisein. This crude concentrate may be prepared by cultivating a strain of the organism Streptomyces griseus which produces L. L. D. active ingredients from which vitamin B12 may be derived as herein described such, for example, as certain streptomycin or grisein producing strains of the organism, on a suitable nutrient medium, adsorbing the active material from the culture broth with a suitable adsorbent material, preferably activated charcoal, eluting the adsorbate with a suitable solvent such as an aqueous solution of pyridine or an alkyl substituted pyridine and concentrating the crude eluate to dryness. Procedures suitable for preparing a crude concentrate which may be utilized as a starting material in our process are disclosed in the application of Frederick A. Kuehl, Jr., and Louis Chaiet, Serial No. 18,848, filed April 3, 1948, which has issued as Patent No. 2,505,053. We have found that such concentrates contain in addition to grisein, L. L. D. active ingredients suitable for deriving vitamin B1: by our process. By our treatment,

1 Claim. (Cl. l6781) l 2 grisein and also streptomycin if present in the starting concentrates used are removed, and our products are free from grisein and streptomycin.

In accordance with one embodiment of our invention, a crude concentrate predominantly composed of Streptomyces griseus elaboration products is extracted with an extraction solvent such as water, or aolower aliphatic water miscible alcohol, preferably methyl alcohol. The selection of the extraction liquid is determined by the adsorbent material to be used in the chromatographic fractionation.

Various adsorbents can be utilized at various stages in the practice of this invention, including activated carbon, activated alumina, and the like. Activated alumina is the preferred adsorbent in the chromatograph step or steps. Aqueous extractions are employed when chromatographic out until its level reaches the top of the adsorbent material in the column. The adsorbent is conveniently supported on a wire screen covered with a layer of sand.

When an alumina column is employed for the chromatograph, the alcoholic, preferably methanolic extract is poured on the upper surface of the adsorbent in the column, and allowed to flow into the adsorbent either by gravity, or under pressure. The column is then developed with additional amounts of the extraction solvent to develop in the column zones of adsorbent ma-. terial containing vitamin B12 in differing amounts and differing degrees of purity, and suitable fractions of the eluate are collected as the zones emerge from the bottom of the column. Those fractions containing sufficient activity as determined microbiologically or by virtue of their pink color are concentrated either separately or combined to a heavy syrup. This concentrate is then mixed with a lower aliphatic alcohol and a miscible non-solvent in which the active substance is insoluble, such as acetone is added. The red flocculent precipitate which forms is recovered.

The red flocculent precipitate may be further purified by dissolving it in a solvent for vitamin B12, such as methyl or ethyl alcohol or water, and precipitating it with a miscible non-solvent for vitamin B12, such as acetone, one or more times.

During such treatment, the solution, if not clear remove insoluble impurities. The number of such precipitations and treatments desirable varies with different batches. A guide to the 1 number of useful precipitations is absence of brownish or yellowish color in the supernatant liquid after precipitation. In some'cases, after .several such treatments, vitamin B12 may be crystallized from a. water solution by adding acetone to the point of incipient turbidity and allowing red needlelike crystals to form.

However, when a crystalline product or a high degree of purification is wanted, it is often desirable to subject the product obtained from the first chromatographic fractionation (either. before or after one or more of the above-mentioned precipitation steps) to a second chromatographic fractionation. This is accomplished by dissolving the amorphous product in a lower aliphatic alcohol. The solution can be added to a second column of activated alumina and the more active fractions of eluate as determined above can be collected and concentrated to dryness. The dry residue may be dissolved in methyl alcohol and acetone added to the solution causing a red precipitate of vitamin B12 to separate. This precipitate may be dissolved in water and acetone added to the solution causing red crystals to separate. The crystals may be redissolved in water and precipitated with acetone several times to remove any impurities that may still remain.

Vitamin B12, obtained in a substantially pure state in accordance with our invention, is a red compound having an approximate composition typified by the following analysis made on two samples dried in vacuo for two hours at 100 C.:

C, 56.35, 56.11; H, 6.72, 6.72; N, 14.51, 14.76; P,-

2.24, 2.27; Co, 4.42, 4.58. Substantially all of the differences between the sum of the percentages given and 100% is believed to be oxygen and no other metals are indicated to be present by spectrum analysis. Based on the foregoing analysis, vitamin B12 has the approximate empirical formula of about C6164H8692Nl4013PCO. Vitamin B12 is substantially soluble in water, methyl alcohol, ethyl alcohol and phenol and substantially insoluble in acetone, ether and chloroform. Vitamin B12 has an apparent partition coeflicient of about 1.46 in the system: 75% toluene 25% ortho-cresol: water. It has been found that vitamin B12 has a partition coefficient of about 1.2 in the system water/benzyl alcohol.

Vitamin B12 crystallizes from suitable solutions such as aqueous acetone as red crystals having the following crystallographic properties: refractive indices 11, 1.619; p, 1.649; 7, 1.659. Vitamin B12 crystals have no definite melting point but darken at about 210-220 C.

The adsorption spectrum of vitamin B12 in aqueous solution is characterized by maxima at 2780 11..

at 3610 A.,

and at 5500 A.,

The spectrum does not change markedly with change of pH; in acid solution the intensity of the 3610 A. band decreases by about 10% and in alkaline solution there are other small changes and the fine structure becomes less marked.

The infrared absorption spectrum was determined with a carefully calibrated Perkin-Elmer 12A Spectrometer. The following absorption maxima were observed:

Wave length in mu (10- cm.)

3.05 S (broad) 8.15 S 4.62 W 8.67 S 5.98 S 9.36 S 6.13 M 10.00 S 6.70 S 10.80 W 7.14 S 11.10 W 7.35 S 11.81 M 7.60 W 12.35 M

ments vary with the individual case and are dependent upon the severity and duration of the pathologic condition and the therapeutic response of the patient. The minimum effective therapeutic dose administered intramuscularly in the form of an aqueous, saline. or isotonic solution is thought to be approximately 1 microgram per day or multiples of this quantity at longer intervals, e. g. approximately 7 micrograms per week.

Various clinical investigations of vitamin B12 therapy in the treatment of macrocytic anemias are reported in the literature including:

Spies, T. D., Stone, R. E., Garcia Lopez, (11., Milanes, F., Lopez Toca, E., and Aramburu, T.,

Thymine, Folic Acid, and Vitamin B12 in Nutritional Macrocytic Anemia, Tropical Sprue, and Pernicious Anemia, Lancet, volume 2, pages 519-522, October 2, 1948; and 7 Hall, B. E., and Campbell, D. 0., Effect of Vitamin B12 on the Hematopoietic and Nervous Systems in Addisonian Pernicious Anemia," Journal of Laboratory and Clinical Medicine, volume 33, page 1646, December 1948.

Vitamin B12 is also capable of increasing the growth rate of chicks. For example, in one experiment the addition of thirty parts per billion of substantially pure vitamin B12 to a basal diet containing purified casein (so-called vitaminfree) as the protein source caused an increase in growth rate such that after sixteen days feeding the average weight of the chicks receiving the supplemented diet was 127 grams, whereas that of the chicks receiving the unsupplemented diet was 99 grams. The preparation of concentrates suitable for chick feeding is dealt with in our above-mentioned pending application Serial No. 38,175.

The activity of the products referred to in this application was determined by a microbiological growth response assay method using Lactobacillus Zactzs Dorner as the test organism. A brief description of the assay method which we employed is given below.

Lactobacillus lactis Dorner has been reported to require two growth factors, T. J. and L. L. D. Medium modifications incorporated in the formula described below, have eliminated therequirement for the T. J. factor, and the assay as presented is specific for L. L. D. This microorganism shows L. L. D. factor response to vitamin B12. Pure crystalline vitamin B12 is used as the able in the American Type Culture Collection.

Stock cultures of the organism are maintained on a growth medium which consists of:

' 1% Difco yeast extract 0.02% tomato juice serum 1% anhydrous dextrose 1.5% agar The medium used for'the inoculum consists of the basal or assay medium to which 1 unit per cc.

of vitamin B12 is added. The inoculum cells arewashed with sterile distilled water and diluted to form a suspension which reads between 90 and 95% light transmission on the Evelyn photometer with a 520 mu filter.

The composition of the assay medium,- double strength, is listed below. 'Ingredients may be conveniently dispensed from stock solutions:

DL isoleucine mg 200 DL alpharalanine mg 200 DL aspartic acid mg 200 DL valine "mg" 200 'DL methionine mg 200 DL glutamic acid mg 200 DL threonine mg 200 DL serine mg 200 DL phenylalanine mg 200 DL leucine mg 200 L histidine mg 200 DL tryptophane -mg 400 L arginine mg 200 Llysine --mg 100 Aminoacetic acid mg 200 Lcystine mg 200 DL norleucine mg 200 L tyrosine mg 200 Dextrose "gm" Sodium acetate gm 6 Fumaric acid gm 0.5 Sodium ethyloxalacetate gm 0.5 Riboflavin mcg.. 200 Calcium pantothenate mcg 200 Thiamin HCl mcg 200 Nicotinic acid ..mcg.. 200 Pyridoxamine mcg 400 Para-aminobenzoic acid mcg 40 Biotin mcg 0.4 MgSOrflHzO "mg" 200 NaCl mg 10 FeSOa'ZHzO mg 10 MIlSO4;4HzO .mg 10 K2HPO4 -4 mg 500 KH2PO4 mg 500 Folic acid mcg 2 Casein hydrolyzate gm 1.0

Water to 500 cc.

The basal medium is prepared by combining the amino acids, then adding dextrose, sodium acetate, fumaric acid, heating to dissolve, and immediately readjusting to pH 7. The sodium ethyloxalacetate and vitamins are then added, dissolved, and the solutions again adjusted to pH 7. Finally, the salts, folic acid and casein hydrolyzate are added, dissolved, and the pH adjusted to 6.6.

A water solution or suspension of the sample to be assayed is diluted so that the solution contains about 0.2 L. L. D. type unit per cc. which is then added to the assay tubes in 0.5, 1.0, 1.5.

- tation.

2.0. and 2.5 cc. amounts. All of the tubes are then adjusted to a volume of 2.5 cc. with water, 2.5 cc. of the basal medium added, and the tubes are finally plugged and sterilized by heating at C. for 13 minutes. After cooling to room temperature, the tubes are inoculated with one drop of the standardized suspension of L. lactis and then incubated at 37 C. for 40 hours. After incubation, the tubes are titrated directly with 0.05 N sodium hydroxide to the blue-green end point of bromthymol blue.

The activity of the sample may then be determined from the standard curve by the amount of sodium hydroxide required.

The standard curve is prepared with crystalline vitamin B12. One milligram of pure crystalline vitamin B12 contains 11 10 units. stock solution is diluted and 0.0, 0.05, 0.1,. 0.2. 0.3. 0.4, 0.6, 0.8, 1.0 and 2.0 units added'to duplicate series of tubes. Water is added to adjust the volume to 2.5- cc. The procedure described above for the samples under assay is followedwith the standard. Typical titration values for the standard series listed above are 1.5, 2.0. 2.7. 4.3, 5.7. 6.6. 7.5, 7.9, 8.0 and 8.4,.respectively, in terms of milliliters of 0.05 -N sodium hydroxide required to neutralize the acid produced per culture of L. lactis.

The following examples illustrate methods of carrying out the present invention, but it is to be understood that these examples are given primarilyby way of illustration and not of limi- Example 1 Aculture broth was prepared by cultivatinga grissein producing strain of the microorganism Streptomyces griseus in a suitable-culture medium. The resulting broth was freed of the propagation organismand other solid material and treated with an adsorbent material such as carbon. The active material was eluted from the adsorbent with an eluting agent such as aqueous pyridine or aqueous a-DlCOliIlB. The eluate was then evaporated to a small volume, diluted with methyl alcohol, and the active concentrate precipitated with ether. The concentrate was then dried. This procedure for the recovery of the active concentrate is disclosed in the application of Frederick A. Kuehl, Jr., and Louis Chaiet, Serial No. 18,848, filed April 3, 1948.

This dried concentrate had a microbiological potency of about 1500 to 2000 L. L. D. units/mg. 750 grams of this dried concentrate was added to about 2.5 liters of dry methyl alcohol. The mixture was stirred for approximately 30 minutes and then filtered. About 2.5 liters of dr" methyl alcohol was then added to the solid residue and the mixture was stirred and filtered. This procedure was repeated with two successive 2.5 liter portions of dry methyl alcohol. The combined filtrates had a total microbiological activity of about 1.2 billion L. L. D. units.

The combined filtrates were chromatographically fractionated by adsorption on about 7.5 kg. of activated alumina wet with methyl alcohol. Methyl alcohol was then passed through "the column and a number of fractions of eluate were collected. These fractions containing high activity (usually at least 10,000 L. L. D. units/ml.) as determined microbiologically and usually indicated by a pink coloration were combined and concentrated at a temperature below 30 C. and a pressure less than atmospheric to about onetenth of the original volume. A small amount of white insoluble material which formed was removed by filtration. The filtrate was then concentrated in vacuo to a heavy syrup. Absolute ethyl alcohol was added to the syrup and the white insoluble precipitate which separated from solution was removed. An equal volume of acetone was then added to the clear red ethyl alcohol solution, causing a red fiocculent precipitate to separate from solution.

The active material precipitated from ethyl alcohol by the addition of acetone was dissolved in approximately 2 ml. of Water and again precipitated in the form of a red oil by the addition of about 12 volumes of acetone. The oil was dissolved in about 1 ml. of water, and the solution treated with acetone until turbidity was observed. Upon standing vitamin B12 separated from the solution in clusters of small red needle-like crystals.

Example 2 A red fiocculent precipitate formed by the addition of acetone to the clear red ethyl alcohol solution secured as in Example 1, was further purified by the following procedure. Upon washing the precipitate with acetone and drying, there was secured a substance having a microbiological potency of about 4 million L. L. D., units/mg. or about 36% vitamin B12. A second crop having similar potency was recovered by the addition to the mother liquor of a second volume of acetone. The further addition of acetone to the mother liquors resulted in the precipitation of a red oil which was recovered. The oil was treated with a mixture of ethyl alcohol and acetone and an amorphous red solid having a microbiological potency of about 1.5 million L. L. D. units/mg. or about 13.5% vitamin B12 was precipitated and recovered.

The several dried acetoneinsoluble fractions were combined, dissolved in approximately 4 ml. of methyl alcohol and the solution was adsorbed on about grams of activated alumina wet with methyl alcohol in a chromatographic column. The column was then developed with methyl alcohol and a dark red band passed through the column. Upon the appearance of the red color in the eluate, successive 10 ml. fractions were taken until the effluent was substantially colorless. The first three fractions, containing the greater part of the red color, were combined and evaporated to dryness at a pressure less than atmospheric and a temperature below 25 C.

The dry residue was dissolved in approximately 2 ml. of methyl alcohol and the solution treated with about 8 volumes of acetone causing a red amorphous precipitate to separate from solution. This precipitate was recovered by centrifugation and reprecipitated in similar manner from a mixture of methyl alcohol and acetone. The red amorphous precipitate consisted of vitamin B12 in a state of about 50% purity (5 million L. L. D. units/mg). A portion of this material (5 me. from a total of 21.8 mg.) was dissolved in approximately 0.5 ml. of water and the solution treated with about 5 ml. of-acetone. Upon standing clumps of red needle-like crystals of vitamin B12, having a microbiological potency of about 11 million L. L. D. units/mg, separated from solution and were recovered.

Example 3 About 1383 grams of a dried concentrate prepared like the starting material. in Example 1 from elaboration products of a grisein producing 8 strain of Streptomyces griseus and having a microbiological potency of about 2000 L. L. D. units/mg. was added to about 16 liters of dry methyl alcohol. The mixture was stirred for approximately 30 minutes and filtered.

The filtrate was adsorbed on about 15 kg.of activated alumina wet with methyl alcohol in the chromatographic column. The column was then developed with methyl alcohol and a number of fraction of eluate were collected.

The chromatograph fractions having the most pronounced microbiological activity were combined and concentrated in vacuo to about onetenth the original volume. The concentrated mixture was filtered and the filtrate further concentrated in vacuo to a heavy syrup. Ethyl alcohol was added to this syrup and the mixture filtered. The filtrate was then treated with sufflcient acetone with stirring to cause complete precipitation of the red material contained therein. The red precipitate was recovered and redissolved in about ml. of methyl alcohol. This solution was then poured slowly with stirring into about 1 liter of acetone. A light red fiocculent material precipitated from the solution and was recovered. Upon washing and drying the precipitate, there was secured 7.8 g. of a red powder, having a microbiological potency of about 65,000 L. L. D. units/mg. or about 0.6% vitamin B12.

It is noted that in this example the vitamin B12 content of the product obtained in one chromatographic step (0.6% vitamin B12) was much less than in the other examples and very much less than need be the case. The example i given to illustrate the fact that our treatment, even when not conducted to obtain maximum potency from the chromatographic treatment, yields a product which is far more potent than any previously known anti-pernicious anemia concentrates with which we are familiar, and which, if desired, may be used for clinical purposes without further concentration.

In general, factors which increase the vitamin B12 content of the product obtained in our chromatographic steps are: (1) increasing the ratio of the weight of the alumina adsorbent to the weight of the solids adsorbed; (2) care in excluding water from the concentrate adsorbed and the alcohol solvent used; (3) care in selecting the most potent fractions.

Examples 4 10.0 grams of concentrate of the type described in the preced ng example having an average potency of 80,000 L. L. D. units/mg. was dissolved in methyl alcohol and the solution adsorbed on about 500 grams of activated alumina wet with methyl 'alcohol in a chromatographic column. The column was then developed with methyl alcohol and separate fractions of eluate collected.

The fractions having pronounced microbiological activity were selected, combined and evaporated to dryness at a pressure less than atmospheric. The residue was extracted with absolute ethyl alcohol and the white substance which failed to dissolve was removed. The red ethyl alcohol solution was treated with an excess of acetone, causing an insoluble red oil to separate. This oil was recovered and dissolved in about 10 ml. of water. The active material, with some accompanying inert matter, was again precipitated as an oil by the addition of excess acetone. The highly active red material was redissolved in about 3 ml. of water and the solution treated with acetone until a faint turbidity was noted. Upon standing, red needle-like crystals of vitamin B12 separated from the solution.

Example 13.5 kg. of ether precipitated concentrate having an L. L. D. activity of about 2000 units/mg. prepared as in Example 1 was extracted three times by stirring for thirty minutes with 13 gal. of methyl alcohol. The combined extracts were chromatographed on 1'7 columns, each six inches in diameter and containing 7.5 kg. of activated alumina wet with methyl alcohol. Each column was developed with methyl alcohol and a series of 1.5 liter eluate fractions was collected as soon as the red color, characteristic of vitamin B12, appeared in the elu-ate. The first three of these eluate fractions from all of the columns were combined and evaporated to approximately 500 ml. at reduced pressure and a temperature below 30 C. The solution was frwd of a white insoluble material by filtration and treated with acetone until the red color was essentially completely precipitated in the form of red oil. The yellowish-brown supernatant liquid was discarded. The red oily residue was redissolved in a, minimum quantity of methyl alcohol and the solution again treated with acetone until substantially all of the red active substance was precipitated. The procedure was repeated until the acetone-methyl alcohol supernatant liquid was substantially free of yellow-brown color. The red oily precipitate was then dissolved in a minimum quantity of methyl alcohol, and the resulting solution poured slowly into volumes of acetone. The red amorphous precipitate which formed was removed. by filtration, washed with acetone, and dried in a vacuum at room temperature. Its L. L. D. potency corresponded to a content of about 5% vitamin B12. The oral administration in a single dose of a 5% vitamin B12 concentrate equivalent to 600 micrograms of vitamin B12 produced a maximal hematological response in one patient; in another, an amount equivalent to 25 micrograms was hematopoietically active when injected intramuscularly in a single dose. This material for injection was prepared by dissolving the vitamin B12 concentrate in a saline solution.

Example 6 A broth was prepared by cultivating a streptomycin producing strain of the microorganism Streptomyces griseus in a suitable culture medium. The resulting broth was freed of the propagation organism and other solid material and contacted with an ion-exchange resin deriving its exchange capacity from carboxylic groups whereby the streptomycin was adsorbed on said resin. The broth from which streptomycin had been removed was then treated with carbon. The active material was eluted from the carbon adsorbate with aqueous pyridine and the eluate evaporated to a small volume and the active concentrate precipitated with ether. The concentrate was dried.

The dry ether-precipitated solid concentrate (1628 g. including 700 g. of filter-aid), which had an L. L. D. activity of 2500 units/mg, was ground to a fine powder and extracted four times by stirring for 30 minutes with 3500 ml. of methyl alcohol and filtering. The combined methyl alcohol extracts were chromatographed on kg. of activated alumina and the column washed with methyl alcohol. As soon as the red color characteristic of vitamin B12 appeared in the cffluent, a series of 3000 ml. fractions was collected.

. 1o The first five of these. containing a total of 673 million L. L. D. units, were combined and concentrated in vacuo at a temperature below 40 C. to ml. The impure vitamin B12 was recovered by pouring this solution into 1200 ml. of acetone; the resulting red precipitate was removed by centrifugation, washed with acetone, and dried at room temperature. Further purification was attained by dissolving the solid in five milliliters of methyl alcohol and adding 25 ml. of acetone. The resulting precipitate was collected by centrifugation, washed with acetone and dried. The yield was 89.8 mg.

The above red solid was dissolved in 3 ml. of methyl alcohol and chromatographed on 4.5 g. of activated alumina. The column was washed with methyl alcohol. When the red color characteristic of vitamin B12 appeared in the eluate, a series of three to four ml. fractions was collected. The first five of these, containing substantially all of the red color, were individually evaporated to dryness, dissolved in about 0.3 ml. of water, and the solution treated with acetone until slightly turbid. On'standing for 24 hours, red needle-like crystals formed in each of the five tubes. The crystals were collected and combined, washed with acetone, and dried. The yield was 14.1 mg. of crystalline vitamin B12. This product was tested and found to be clinically active.

The various concentrates produced by the methods herein described which possess L. L. D. activity of 65,000 units/mg, or more, are all suitable for clinical use, being free from harmful impurities. They are the subject matter of our copending application filed herewith.

The present application is directed to vitamin B12 itself, a new product which it is believed applicants were the first to discover or produce by any method. For therapeutic use, it may be administered parenterally in the form of any suitable therapeutic solution, such as aqueous, saline, or isotonic solutions, and the concentration may be varied over wide limits to suit the needs of each particular case.

Various features of novelty described but not claimed herein are the subject matter of our copending application, Serial No. 110,222.

Modifications may be mad in carrying out the present invention without departing from the spirit and scope thereof and the invention is to be limited only by the appended claim.

We claim:

The compound vitamin B12. anorganic substance containing cobalt, together with carbon, nitrogen, hydrogen, oxygen, and phosphorus, said compound being a red crystalline substance soluble in water, methyl and ethyl alcohol and phenol, and insoluble in acetone, ether and chloroform, and exhibiting strong absorption maxima at about 2780 A 3610 A. and 5500 5., and an L. L. D. activity of about 11,000,000 L. L. D. units per milligram.

EDWARD L. R'ICKES. THOMAS R. WOOD.

REFERENCES CITED The following references are of record in the tile 0! this patent:

Subbarow: Vitamins and Hormones, vol. 111 (1945).pa e 2'70.

Daki'n: Jour. of Biol. Chem, vol. 115, pp. 771 to 787 (1936).

Science. vol. 107, Apr. 16, 1948. pp. 396 to 398. 

